Automated system and method for harvesting and multi-stage screening of plant embryos

ABSTRACT

A method and system for automatically harvesting and screening plant embryos in multiple stages to identify those embryos that are suited for incorporation into manufactured seeds are provided. The method includes generally three steps. First, plant embryos are automatically sorted according to their rough size/shape and also singulated into discrete embryo units, for example by vibrational sieving. Second, the sorted and singulated plant embryos are classified using a first classification method. For example, each embryo may be imaged by a camera and the image is used to ascertain the embryo&#39;s more precise size/shape. Third, for those embryos that have passed the first classification method, a second classification method is applied. For example, a pre-developed classification algorithm to classify embryos according to their putative germination vigor may be applied to the same image used in the first classification method, to identify those embryos that are likely to germinate.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. ProvisionalApplication No. 60/509,070, filed Jun. 30, 2003.

FIELD OF THE INVENTION

The invention is directed generally to manufactured seeds and, moreparticularly, to a method and system for automatically harvesting andscreening mass-produced plant embryos in multiple stages to identifythose embryos that are suited for incorporation into manufactured seeds.

BACKGROUND OF THE INVENTION

Reproduction of selected plant varieties by tissue culture has been acommercial success for many years. The technique has enabled massproduction of genetically identical selected ornamental plants,agricultural plants and forest species. The woody plants in this lastgroup have perhaps posed the greatest challenges. Some success withconifers was achieved in the 1970s using organogenesis techniqueswherein a bud, or other organ, was placed on a culture medium where itwas ultimately replicated many times. The newly generated buds wereplaced on a different medium that induced root development. From there,the buds having stems and roots were planted in soil.

While conifer organogenesis was a breakthrough, costs were high due tothe large amount of handling needed. There was also some concern aboutpossible genetic modification. It was a decade later before somaticembryogenesis achieved a sufficient success rate so as to become thepredominant approach to conifer tissue culture. With somaticembryogenesis, an explant, usually a seed or seed embryo, is placed onan initiation medium where it multiplies into a multitude of geneticallyidentical immature embryos. These can be held in culture for longperiods and multiplied to bulk up a particularly desirable clone.Ultimately, the immature embryos are placed on a development mediumwhere they grow into somatic analogs of mature seed embryos. As used inthe present description, a “somatic” embryo is a plant embryo developedby the laboratory culturing of totipotent plant cells or by inducedcleavage polyembryogeny, as opposed to a zygotic embryo, which is aplant embryo removed from a seed of the corresponding plant. Theseembryos are then individually selected and placed on a germinationmedium for further development. Alternatively, the embryos may be usedin artificial seeds, known as manufactured seeds.

There is now a large body of general technical literature and a growingbody of patent literature on embryogenesis of plants. Examples ofprocedures for conifer tissue culture are found in U.S. Pat. Nos.5,036,007 and 5,236,841 to Gupta et al.; U.S. Pat. No. 5,183,757 toRoberts; U.S. Pat. No. 5,464,769 to Attree et al.; and U.S. Pat. No.5,563,061 to Gupta. Further, some examples of manufactured seeds can befound in U.S. Pat. No. 5,701,699 to Carlson et al., the disclosure ofwhich is hereby expressly incorporated by reference. Briefly, a typicalmanufactured seed is formed of a seed coat (or a capsule) fabricatedfrom a variety of materials such as cellulosic materials, filled with asynthetic gametophyte (a germination medium), in which an embryosurrounded by a tube-like restraint is received. After the manufacturedseed is planted in the soil, the embryo inside the seed coat developsroots and eventually sheds the restraint along with the seed coat duringgermination.

One of the more labor intensive and subjective steps in theembryogenesis procedure is the selective harvesting from the developmentmedium of individual embryos suitable for germination (e.g., suitablefor incorporation into manufactured seeds). The embryos may be presentin a number of stages of maturity and development. Those that are mostlikely to successfully germinate into normal plants are preferentiallyselected using a number of visually evaluated screening criteria. Askilled technician evaluates the morphological features of each embryoembedded in the development medium, such as the embryo's size, shape(e.g., axial symmetry), cotyledon development, surface texture, color,and others, and manually plucks desirable embryos out of the developmentmedium with a pair of tweezers. The plucked desirable embryos are thencarefully laid out on a tray in a two-dimensional array for furtherprocessing. This is a highly skilled yet tedious job that is timeconsuming and expensive. Further, it poses a major production bottleneckwhen the ultimate desired output will be in the millions of plants.

It has been proposed to use some form of instrumental image analysis forembryo selection to supplement or replace the visual evaluationdescribed above. For example, PCT Application Serial No. PCT/US00/40720(WO 01/13702 A2) discloses an embryo delivery system for manufacturedseeds including an imaging camera, which acquires and digitally storesimages of embryos. The images are then sent to a computer, whichclassifies the embryos according to their desirability (i.e., likelihoodto germinate and grow into normal plants) based on predeterminedparameters (axial symmetry, cotyledon development, surface texture,color, etc.) using a classification method disclosed in PCT ApplicationSerial No. PCT/US99/12128 (WO 99/63057). Those embryos that areclassified as desirable are thereafter removed by mini-robotic pick andplace systems and inserted into manufactured seeds. The disclosure ofthese two PCT applications is hereby expressly incorporated byreference.

While instrumental imaging analysis and subsequent automatic insertionof desirable embryos into manufactured seeds have been successful inincreasing the efficiency of the embryogenesis procedure, there has notbeen a complete automated process of harvesting embryos, e.g., removingembryos from a development medium, sorting embryos according to theirsize/shape and singulating them into discrete units (e.g., by removingany undesirable tissues or other debris), and classifying them accordingto their desirability for incorporation into manufactured seeds. Inother words, there has not been an automated process that could replacethe current manual operation of plucking desirable embryos out of adevelopment medium and placing them in an array suitable for furthermaturation treatments. The present invention is directed to providing acomplete automated process of harvesting somatic embryos, which couldreplace the current manual operation.

SUMMARY OF THE INVENTION

The present invention provides a method and system for automaticallyharvesting plant embryos. According to one aspect, the automaticharvesting method of the invention screens plant embryos in multiplestages to identify those embryos that are suited for incorporation intomanufactured seeds, i.e., those embryos that are both physically fit forincorporation into manufactured seeds (not too big, not too small, nottoo bent, etc.) and also qualitatively determined to be likely togerminate and grow into normal plants. The automatic harvesting methodincludes generally three steps. First, plant embryos are automaticallysorted according to their size/shape and also singulated into discreteembryo units. For example, the embryos may be washed off from adevelopment medium (e.g., from a development pad) using aqueous liquidand sieved through a porous material. During sieving, the embryos may befurther sprayed with aqueous liquid to facilitate removal and washingaway of any undesirable material, such as undersized embryos, tissues,and residual embryonal suspensor masses (ESM), through the holes of theporous material. In one preferred embodiment, the porous material isformed as a moving porous conveyor belt so that the embryos being sortedand singulated are simultaneously transported to the subsequentclassification stage. Second, the sorted and singulated plant embryosare classified using a first classification method. For example, each ofthe embryos may be imaged by a camera and the image is used to ascertainthe embryo's size/shape. Those embryos within a predefined size/shaperange are considered to have passed the first classification method.Third, at least for those embryos that have passed the firstclassification method, a second classification method is applied tofurther select those embryos desirable for incorporation intomanufactured seeds. For example, a pre-developed classificationalgorithm to classify embryos according to their putative germinationvigor (i.e., likelihood of successful germination) may be applied to thesame image used in the first size/shape classification method, toidentify those embryos that are likely to germinate. The embryos thathave passed both the first and second classification methods areidentified as suitable for incorporation into manufactured seeds.

According to one aspect, the first and second classification methods arecarried out along a classification conveyor belt while the sorted andsingulated embryos are transported thereon. In some classificationmethods, it is preferred that the embryos are generally arranged in asingle file on the classification conveyor belt. Various means forachieving the single file configuration are proposed. For example, theclassification conveyor belt may be arranged generally perpendicularlyto the porous conveyor belt on which the embryos are sorted andsingulated. According to this configuration, the sorted and singulatedembryos transported to the end of the porous conveyors may droptherefrom by gravity onto the classification conveyor belt to generallyform a single file thereon. To achieve sufficient spacing between theembryos in a single file, the initial rate of washing off embryos from adevelopment medium onto the porous conveyor belt or the speed of theporous conveyor belt may be adjusted, perhaps based on the actual rateof embryos being dropped from the porous conveyor belt onto theclassification conveyor belt as observed by a suitable optical scanningsystem.

According to another aspect, the method further includes the step ofautomatically removing those undesirable embryos that have failed thefirst or second classification method from the classification conveyorbelt. For example, a computer-controlled air or liquid jet may be usedto eject undesirable embryos. The precise timing of the jet activationcan be computer controlled because the position of each undesirableembryo is precisely known based on the firing time of the camera thathas imaged each embryo and the speed of the classification conveyorbelt.

According to yet another aspect, the method further includes the step ofautomatically removing those desirable embryos that have passed both thefirst and second classification methods from the classification conveyorbelt. In one embodiment, the desirable embryos are automaticallytransferred onto a receiving surface in an evenly spaced array, suitablefor various further maturation treatments. For example, the receivingsurface may be provided by a tray mounted on a motorized platformconfigured to adjust the position of the tray relative to theclassification conveyor belt. By adjusting the position of the traybased on the known position of each desirable embryo as it is droppedfrom the classification conveyor belt, the desirable embryos may bereceived on the tray in an evenly spaced two-dimensional array.

According to yet another aspect, the method may include a step ofautomatically removing those desirable embryos that have passed one ormore initial classification methods from a conveyor belt. For example, amini-robotic system may be used to pick up those embryos determined tobe within an acceptable size/shape range and to precisely place them inan evenly spaced two-dimensional array on a receiving tray. At thistime, the embryos may be oriented uniformly, for example, with theircotyledon ends facing the same direction. The properly oriented andprecisely spaced apart embryos in a tray may then be forwarded toreceive further treatments, for example, drying and subsequent furtherclassification methods. Thereafter, these properly oriented and spacedapart embryos in a tray can be readily transferred and inserted intomanufactured seeds which, advantageously, may be arranged in acorrespondingly evenly spaced array.

Classifying the embryos in multiple stages achieves efficient screeningof embryos. For example, by classifying embryos using a relatively lesssophisticated and less time-consuming classification method first, onecan reduce the number of embryos to be forwarded to the secondclassification method that is more sophisticated and moretime-consuming. Thus, by carefully selecting suitable classificationmethods to be combined, one can achieve increasingly selective anddiscriminating classification of embryos in a time efficient manner.Also, the present invention offers a complete automated process ofharvesting somatic embryos, including sorting and singulating embryos(starting with removing the embryos from a development medium),classifying the sorted and singulated embryos according to theirputative germination vigor, and further arranging those embryosclassified as desirable in a manner suitable for further maturationtreatments, e.g., in an evenly spaced two-dimensional array on a tray.Thus, an automated harvesting method and system of the present inventioncould replace the current manual operation of plucking desirable embryosfrom a development medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A schematically illustrates a system for automated harvesting andmulti-stage screening of plant embryos, in accordance with the presentinvention;

FIG. 1B schematically illustrates an alternative system for automatedharvesting and multi-stage screening of plant embryos, in accordancewith the present invention;

FIG. 2 is a flowchart illustrating an overall flow of a method forautomatically harvesting and screening embryos in multiple stages, inaccordance with the present invention; and

FIGS. 3A and 3B illustrate alternative methods of automatically sortingand singulating embryos, according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention pertains to a method and system for automaticallyharvesting and screening mass-produced embryos, such as somatic embryos,preferably in multiple stages of increasing complexity to identify thoseembryos that are suited for incorporation into manufactured seeds. Asused herein, an embryo suited for incorporation into a manufactured seedmeans an embryo that is both biochemically matured (i.e., likely togerminate and grow into a normal plant) and morphologically orphysically suited for incorporation into a manufactured seed (i.e.,having a size/shape appropriate to be included in a manufactured seed).

Referring to FIG. 1A, in a Development Room 10, somatic embryos havebeen developed from embryonal suspensor masses (ESM) andsupported/suspended in or on a development surface 16. A developmentsurface may be provided by a development pad, as illustrated, or may beprovided by any other suitable development medium including a gel-formmedium, or may further be provided by an intervening surface that isplaced on a development medium such as a stainless steel mesh. While thefollowing description illustrates a case in which a development pad isused to provide a development surface, it should be understood that adevelopment surface, as used in the present application, refers to anysurface that supports or suspends embryos that are developed from ESM.

Methods of developing somatic embryos are known and described in variouspublications, as discussed in the background section above. Desirableembryos are to various degrees attached to and embedded in suspensortissues and residual underdeveloped ESM (or culture material) in the pad16, together with incompletely developed embryos, abnormally formedembryos, undersized or oversized embryos, and other pieces of non-embryoplant material. The embryos suspended in a development pad 16 areforwarded to a Harvesting and Classification Room 12, in which theembryos (embedded in the culture material) are removed from thedevelopment pad and further automatically sorted, singulated, andclassified according to their desirability. Classification may becarried out using multiple stages of increasingly sophisticated and yettime-consuming classification methods, to achieve progressively higherselection accuracy and operational efficiency. Those embryos that areclassified as desirable are thereafter forwarded to receive furthermaturation treatments, for example, to a Post Development Treatment Room(Drying Room in the illustrated embodiment) 14 to be dried for storageand subsequent incorporation into manufactured seeds. The presentinvention is generally directed to the automated process of harvestingand classifying embryos, which occurs in the Harvesting andClassification Room 12. It is contemplated that the harvesting andclassification are carried out preferably in a humid clean roomconditioned to sustain viability of the embryos being processed.

Referring additionally to FIG. 2, a method of the present invention inone embodiment includes generally four steps. First, referring to step“A” in FIGS. 1A and 2, embryos are washed off the development surface(e.g., development pad surface) 16 using pressure-controlled sprays ofaqueous liquid (e.g., isotonic nutrient solution) from a suitablyarranged nozzle 18. This washing off process separates the culturematerial including embryos from the development surface 16, but at leastsome of the embryos remain embedded in or attached to suspensor tissues,residual underdeveloped ESM, and other materials at this point. Asillustrated, the development surface 16 may be placed on an inclinedsurface 20 to facilitate washing off and removal of the culture materialincluding embryos via gravity toward a reservoir 22 (or ahydrocyclone-type separator). The bottom 26 of the reservoir 22 has anelongate opening (slit) extending generally perpendicularly to thedirection of a conveyor belt 24 and extending substantially throughoutthe width of the conveyor belt 24, so that the embryos (embedded in orattached to suspensor tissues or residual ESM) are placed onto theconveyor belt 24 in a generally spread or spatially uniform manner.Alternatively, a flow of liquid-dispersed embryos from the reservoir 22can be regulated by various other means, such as constriction, flow pathlength adjustment, etc., to place the embryos on the conveyor belt 24 ina regulated, spatially uniform manner.

Next referring to step “B,” the conveyor belt 24 is formed of a porouscontinuous belt 28 driven by a suitable motor (not shown), which sortsand singulates the embryos by sieving. As used herein, “sorting andsingulating” means rudimentarily classifying embryos according to theirsize/shape and also separating the embryos into discrete units, forexample by separating embryos apart and also by removing any undesirablematerials from each embryo. For example, sieving by the porouscontinuous belt 28 achieves both sorting and singulation by causing anyundersized material, such as undersized embryos and debris, to dropthrough its holes.

Specifically, while on the porous continuous belt 28, the embryosperhaps still embedded in suspensor tissues and residual ESM may befurther sprayed with aqueous liquid from a second nozzle 30 to cause theembryos (and other adhering materials) to be further dispersed in theaqueous liquid. The liquid spray causes adhering suspensor tissues andresidual ESM to be detached from the embryos and washed away and droppedthrough the porous belt 28. Any undersized or incompletely formedembryos will also be dropped through the porous belt 28. In oneembodiment, the conveyor belt 24 may be of a vibrating type, as wellknown in the conveyor belt technology field, to further facilitate thesorting and singulation process. Any material dropped through the porousbelt 28 may be collected in a waste receptacle 32 placed underneath theporous belt 28. Optionally, a second coarser porous belt (not shown) maybe provided in series with the first conveyor belt 24 having the firstporous belt 28, perhaps prior to the first porous belt 28, to carry awayany oversized embryos and other oversized pieces of material. Thus, onlythose mostly singulated embryos of generally desired size and/or shape,which are more or less free of suspensor tissue and other fine plantmaterial, remain on the first porous belt 28. By adjusting the mesh(hole) size/shape of the porous belt 28 (and of any other additionalporous belts), only those embryos within a desirable size/shape rangecan be selected. It should be noted that, alternatively to the one ormore porous conveyor belts described above, one or more sieves of wireor other mesh, for example, vibrating inclined sieves, may be used,although the use of porous conveyor belt(s) is preferred because theysieve and transport (to the next stage) embryos at the same time.

As described above, during steps “A” and “B”, the heterogeneous milieu(containing, e.g., acceptable quality embryos, unacceptable embryos,suspensor tissues, residual ESM, and other plant material) is dispersedin aqueous liquid and subjected to separation of components by physicalforces (e.g., by sieving) that act differently on the components basedon their physical properties (mass, size, shape, specific gravity, dragcoefficient, wettability, etc.). As a result, fine plant material andembryo-adhering suspensor tissues are removed, with reduction in amountof any other undesirable components, to produce a population comprisingmostly singulated embryos substantially free of suspensor tissues.

After the spray-assisted sieving process, referring to step “C,” at theend of the first conveyor belt 24, the sorted and singulated embryos aredropped by gravity onto another conveyor belt, or a classificationconveyor belt 34. The classification conveyor belt 34 is arrangedgenerally perpendicularly to the first conveyor belt 24 so that thedropping embryos will generally form a single file 36 along the lengthof the selection conveyor belt 34 suitable for subsequent imaging. Incase the embryos tend to stick to the first conveyor belt 24 and cannotbe easily dropped, the separation of the embryos from the first conveyorbelt 24 may be assisted by various means. For example, the embryoremoval may be assisted by an air/liquid jet (e.g., a gentle squirt ofnutrient solution or puff of air-not shown) suitably arranged beneaththe porous belt 28 near the end 35 of the first conveyor belt 24, or afine vibrating wire placed perpendicularly to and just above the firstconveyor belt 24 near the end 35, so as to break the surface tension andknock the embryos off the first conveyor belt 24. Alternatively, a dryer(not shown) may be arranged adjacent to the first conveyor belt 24 todry off the embryos as they move down the first conveyor belt 24.

For the purpose of subsequent imaging, the embryos are sufficientlyspaced apart from each other on the classification conveyor belt 34. Toachieve sufficient spacing between the embryos in a single file 36, theinitial rate of washing off the embryos from the development surface 16may be adjusted. Also, the configuration of the reservoir 22 (or ahydrocyclone-type separator) may be adjusted, as discussed above, toachieve controlled dispensing of the embryos onto the first conveyorbelt 24 and hence controlled dropping of the embryos from the firstconveyor belt 24 onto the classification conveyor belt 34. While thereservoir 22 is illustrated to be positioned upstream of the sprayedsieving process in FIG. 1A, it should be understood that the reservoir22 may be located downstream from the sprayed sieving process, near theend 35 of the first conveyor belt 24, so as to receive and controllablydrop the embryos from the first conveyor belt 24 onto the classificationconveyor belt 34. As yet another example, an electronic embryo positionmapper (not shown) controlled by a computer 40 may be positioned nearthe end 35 of the first conveyor belt 24 downstream of the sprayedsieving process. The embryo position mapper consists of a suitableoptical sensor and detector combination to determine the position ofeach embryo as it is carried on the first conveyor belt 24. The computer40, based on the positional information received from the embryoposition mapper, continuously adjusts the belt speed of the firstconveyor belt 24 and/or the classification conveyor belt 34 so as toachieve a uniform dropping rate of the embryos from the first conveyorbelt 24 onto the classification conveyor belt 34. Any of the methodshereinabove described may be combined together. For example, the embryoposition mapper positioned near the end 35 of the first conveyor belt 24may be used to control the initial washing off rate of the embryos fromthe development surface 16. Also, any other methods for achievingsufficient spacing between the embryos, as they are placed onto thefirst conveyor belt 24, as will be apparent to one skilled in the art,may be used.

In one alternative embodiment, the single file configuration preferredfor imaging purposes may be obtained by utilizing the flow ofliquid-dispersed embryos along a pipe. Specifically, referring to FIG.3A, embryos are washed off the development surface 16 using aqueousliquid from a suitably arranged nozzle 18 and placed into a reservoir22, as with the embodiment illustrated in FIG. 1A. The reservoircommunicates with a pipe 21 having a properly chosen diameter, throughwhich the liquid dispersed embryos still entangled with other tissues,ESM, and plant debris, flow, preferably at a predefined controllablevelocity, and exit onto the porous conveyor belt 25. In one embodiment,the pipe 21 is clear so that an optical scanner 23 arranged along theclear pipe 21 can observe the flow therethrough to provide feedback tothe initial rate of washing off the embryos from the development mediumto ensure a desired level of spacing between materials (e.g., embryos)passing through the pipe 21. As before, the conveyor belt 25 is porous,at least in its upstream portion, so that any undersized embryos andother fine materials (suspensor tissues, residual ESM, etc.) fallthrough the belt 25, as further facilitated by an aqueous liquid sprayfrom the nozzle 30. As illustrated, because the culture stream dispensedfrom the pipe 21 is generally lined up on the porous conveyor belt 25,the embryos remaining on the belt 25 after the sprayed sieving processare already in a single file configuration 34. Thus, the embryos maycontinue directly for further classification on the same conveyor belt25, for example, for image acquisition by a camera 38 and subsequentselective removal of undesirable embryos by an ejector 42, bothcontrolled by the computer 40. The embryos eventually drop from the endof the conveyor belt 25 onto a receiving tray 54, as will be more fullydescribed below.

As a further alternative method of achieving the single fileconfiguration, referring to FIG. 3B, a flow cytometer may be used tosort and singulate embryos. In this embodiment, the embryos are washedoff the development surface 16 using aqueous liquid from a suitablyarranged nozzle 18 and placed into a reservoir 22, as with the previousembodiments, and thereafter travel through a clear pipe 21. A flowcytometer (or cell separator) 27, which is well known in the art, isarranged along the clear pipe 21 to observe and separate the desirableembryos from other materials such as undersized/oversized embryos,suspensor tissues, and residual ESM. Briefly, the flow cytometerdifferentiates different cells transported in liquid based on the cellproperties as observed by optical sensors 27 a, and furtherelectrostatically sorts (separates) the cells using deflectors 27 bbased on ink jet technology, i.e., by deflecting selectively chargedliquid droplets containing the targeted cells. In the illustratedembodiment, the flow cytometer 27 is used to sort and separate (branchoff) embryos 31 that meet the predefined size/shape criteria onto theclassification conveyor belt 34, while other materials 33, such asundersized or oversized embryos and suspensor tissues and residual ESM,drop into a rejects receptacle 29. Therefore, the flow cytometer 27 notonly sorts and singulates the embryos, but also places them in agenerally single file on the classification conveyor belt 34.

Referring back to FIGS. 1A and 2, in step “C”, the sorted and singulatedembryos preferably placed in a single file and spaced sufficiently farapart from each other on the classification conveyor belt 34 areclassified according to their desirability. For example, each of theembryos may be imaged by a camera 38 placed adjacent to (e.g., above)the classification conveyor belt 34. The image of each embryo istransmitted to the computer 40 to be analyzed and classified accordingto one or more high-speed algorithms.

In one embodiment, each image (monochromatic or in color) of an embryois analyzed in two steps. First, referring to FIG. 2, block 46, asuitable algorithm is used to identify those embryos that do not meetbasic size and shape criteria to be incorporated into manufacturedseeds. Second, referring to block 48, a pre-developed classificationmodel, such as those disclosed in PCT Application Serial No.PCT/US99/12128 (WO 99/63057) discussed above, is applied to theremaining embryos (i.e., the embryos that have met the first size/shapecriteria) to identify those embryos having a lower probability ofgerminating (lacking in germination vigor). Briefly, a suitableclassification model can be developed based on a sample population ofembryos for which images and actual germination data have been obtained.In a preferred example, a pre-developed classification model to classifyembryos according to their putative germination vigor is applied to thesame image used in the first size/shape classification step, so that asingle image can be used in both of the first and second classificationsteps. Referring additionally to FIG. 1A, the programs (algorithms) thateffect the actual classification and other evaluation of the embryosbased on the images produced by the camera 38, e.g.; pre-developedclassification models, are stored in the computer 40.

Those embryos rejected either by the first classification step as notmeeting the size/shape criteria (block 46) or by the secondclassification step as not likely to germinate (block 48) may thereafterbe ejected from the classification conveyor belt 34, for example, by aprecisely timed air/liquid jet 42 controlled by the computer 40 into awaste receptacle 44. The precise timing of the jet activation can becomputer controlled because the position of each undesirable embryo isprecisely known based on the firing time of the camera 38 that hasimaged each embryo and the known speed of the classification conveyorbelt 34. The use of an image-actuated precision jet to removeundesirable materials from a conveyor belt is well known in the foodindustry, for example to sort foods based on their visualcharacteristics. After undesirable embryos have been removed, only thoseembryos that have passed both the first and second classification stepsremain on the classification conveyor belt 34. Alternatively, theejector 42 may be configured to remove desirable embryos from theclassification conveyor belt 34 onto another location, such as anotherconveyor belt or a harvest chamber, for further maturation treatments,as will be apparent to one skilled in the art.

The embryo classification step “C” may include further steps or stagesof data acquisition and classification/screening operations. Forexample, after the two-step camera image analysis (blocks 46 and 48 inFIG. 2) is carried out as described above, the embryos may undergo afurther imaging analysis (e.g., block 50) or a spectroscopic analysisusing IR, NIR, or Raman spectroscopy (block 52), as will be more fullydescribed below, before undesirable embryos are removed from theselection conveyor belt 34. Alternatively, after the two-step cameraimage analysis described above is completed and any undesirable embryosare ejected, the remaining desirable embryos may be placed in a tray anddried for storage purposes (step “D”), and thereafter (at a later time)undergo further stages of data acquisition and classification, or the“secondary” classification step “E”, as will be more fully describedbelow. In other words, in accordance with the present invention, theembryos may undergo any number of classification stages, and further,not all of the classification stages need to occur at the same time.

The camera 38 may be of any suitable type as will be apparent to oneskilled in the art, either monochromatic or color, though preferably adigital camera containing a charge-coupled device (CCD) linked to adigital storage device is used so as to permit subsequent digitalprocessing of the acquired image. Further, the camera 38 may be asingle-view camera (e.g., taking only the top view of each embryocarried on the classification conveyor belt 34) or a multiple-viewcamera (e.g., taking the top view, side view, and end view of eachembryo). To acquire multiple views of an embryo, one camera may be movedinto multiple positions, or multiple cameras may be used. However,preferably, a method and system for simultaneously imaging multipleviews of an embryo using a single camera and suitably arrangedreflective surfaces (e.g., prisms) may be used so as to shorten the timeand operation required to obtain multiple views. Such a method andsystem for simultaneously imaging multiple views of an embryo aredisclosed in a copending U.S. patent application, filed concurrentlyherewith, titled “Method and System for Simultaneously Imaging MultipleViews of a Plant Embryo” , which is explicitly incorporated herein byreference. A classification model algorithm may then be applied to eachof the multiple views of an embryo to classify the embryo according toits putative germination vigor.

Additionally or alternatively, during the embryo classification step“C”, an apical dome located at the cotyledon end of a plant embryo maybe three dimensionally imaged and analyzed to determine the embryo'sgerminant vigor (i.e., potential for rapid epicotyl development aftergermination). (See FIG. 2, block 50.) Because the apical dome is wheremost plant cells that produce the plant body are formed, it has beendetermined that the dome's morphological features (size, shape, etc.)are reliable indicators of the embryo's tendency for rapid growth aftergermination. In other words, the three-dimensional information of theapical dome of an embryo can be used as an input to a classificationmodel algorithm to further classify the embryos according to theirdesirability. Some methods of three-dimensionally imaging an apical domeof a plant embryo can be found in a copending U.S. patent application,filed concurrently herewith, titled Method and System forThree-Dimensionally Imaging an Apical Dome of a Plant Embryo, which isexplicitly incorporated herein by reference.

Further additionally or alternatively, during the embryo classificationstep “C”, an embryo may be analyzed using a spectroscopic analysismethod, such as IR spectroscopy, NIR spectroscopy, or Ramanspectroscopy. (See FIG. 2, block 52). The classification modelsdisclosed in PCT Application Ser. No. PCT/US99/12128 (WO 99/63057)discussed above, may be applied to any absorption, transmittance, orreflectance spectra of embryos, to further qualitatively classify theembryos according to their chemical composition. Briefly, aspectroscopic analysis permits identification of chemistry of eachembryo and thus identification of targeted chemical(s) or analytes in anembryo. Embryos that are biochemically mature and likely to germinateare known to include certain levels of targeted chemicals or analytes,such as sugar alcohols. Thus, spectroscopic analysis of embryos is areliable method of qualitatively identifying biochemically matureembryos. Some methods of spectroscopically analyzing and classifyingembryos using NIR spectroscopy are disclosed in PCT Application SerialNo. PCT/US99/12128 (WO 99/63057) discussed above. Further, a method ofassessing embryo quality using Raman spectroscopy is disclosed in acopending U.S. patent application, filed concurrently herewith, titled“Method for Classifying Plant Embryos Using Raman Spectroscopy” which isexplicitly incorporated herein by reference. As used herein,spectroscopic analysis encompasses the analysis of an image taken in oneor more specific spectral bands, commonly known as multi-spectralimaging (or chemical imaging, chemical mapping).

It should be noted that other imaging or spectroscopic technologies todetermine the biochemical composition or morphological structure of anembryo may be used additionally or alternatively to any of theclassification methods described above. As new imaging or spectroscopictechnologies emerge or mature, these technologies can be readilyincorporated into the present method of automated harvesting andmulti-stage screening of plant embryos. For example, Teraherz rays(T-rays) may be used to spectroscopically image a plant embryo todiscern its chemical and physical compositions. As a further example,fluorescent labeling technology, such as the quantum dots technologydeveloped by Quantum Dot Corporation of Hayward, Calif., may be used todetect specific compounds and also to track biological events within aplant embryo. Still further, cosmic rays may be utilized to measure thedensity of an embryo. As will be apparent to one skilled in the artbased on these examples, any other technologies that could determine thebiochemical or morphological (structural) properties of a plant embryo,based on the use of a broad spectrum of electromagnetic radiation, maybe used in accordance with the present invention.

It is noted that the method described hereinabove screens or classifiesembryos in multiple stages, first by sieving based on rudimentarysize/shape criteria (step “B”) then by increasingly sophisticated andhence generally time-consuming means during step “C”, such as animage-based size/shape analysis (block 46), image-based classificationmodel analysis (block 48), image-based apical dome analysis (block 50),and spectra-based chemical analysis (block 52). It should be understoodthat more classification methods may be added as further additionalscreening criteria are developed. For example, a method of determiningthe disease resistance of an embryo may be developed using some sensor.Then, a classification stage to classify embryos based on thedisease-resistance criteria may be added to further refine the overallclassification process. As more screening criteria are developed andtheir corresponding classification methods incorporated into the presentmethod, the method will be able to identify those embryos that arehighly likely to grow into plants that are strong, healthy, and havevarious other desirable characteristics.

It is contemplated that only those embryos that have passed the previousclassification stage will be forwarded to the subsequent screening stageso that a lesser number of embryos need to be evaluated by a laterscreening stage of perhaps increasing sophistication and complexity,since complex screening stages tend to be more time consuming. However,in some situations two or more screening stages may be carried out inparallel, substantially simultaneously. For example, when multiple views(e.g., the top view, the side view, and the end view) of an embryo aretaken and analyzed according to a classification model (block 48), oneof the views (e.g., the cotyledon end view containing three-dimensionalinformation of an apical dome) may be simultaneously analyzed in depthto ascertain the morphological features of the embryo's apical dome(block 50).

Still referring to FIGS. 1A and 2, in step “D”, at the end of theclassification conveyor belt 34, those desirable embryos remaining onthe conveyor belt 34 are dropped by gravity (perhaps assisted by anair/liquid jet-not shown) onto a tray (or pad, or any suitable surface)54 mounted on a two-dimensional drive system (or motorized platform) 56,which is also controlled by the computer 40. The drive system 56two-dimensionally (or perhaps three-dimensionally) adjusts the positionof the tray 54 relative to the end of the classification conveyor belt34 so as to receive embryos dropping therefrom into an evenly spacedarray (e.g., a two-dimensional array). The positioning of the tray 54relative to the end of the classification conveyor belt 34 is determinedbased on the precisely known position of each embryo on theclassification conveyor belt 34 according to the firing time of thecamera 38 and the speed of the conveyor belt 34. Thus, even a somewhatirregularly spaced linear array of desirable embryos on theclassification conveyor belt 34 can be transformed into an evenly spacedtwo-dimensional array on the tray 54. The construction and operation ofthe drive system 56 should be apparent to one skilled in the art andthus need not be described in detail here. The tray 54, perhapscontaining 100 plus embryos arranged in an evenly spaced array, maythereafter be forwarded to receive further maturation treatments. Forexample, the tray 54 may be forwarded to the Post Development TreatmentRoom (Drying Room in the illustrated embodiment) 14 to dry the embryosfor storage and for subsequent incorporation into manufactured seeds.

Additionally, referring specifically to FIG. 2, in step “E”, the embryosplaced and dried in the tray 54 may undergo a further, secondary seriesof classification stages prior to incorporation into manufactured seeds.The embryos may be rehydrated prior to the secondary series ofclassification stages, or may remain desiccated during one or more ofthe secondary series of classification stages, depending on eachapplication. As with the previous classification step “C”, the secondaryclassification step “E” may also include one or more classificationstages of increasing sophistication and complexity to achieveprogressively higher selection accuracy and operational efficiency.Because only a relatively small number of embryos, having passed theprevious classification step “C”, are remaining at this time, moresophisticated and thus time-consuming classification methods can becarried out, such as the multi-view color imaging analysis using aclassification model (block 54), an apical dome analysis (block 56), ora spectroscopic analysis, perhaps also using multiple views (block 58).It is also contemplated that when robotic pick and place systems areused to automatically pick up and insert embryos into manufacturedseeds, some digital imaging may be required to ascertain the position ofeach embryo for that purpose, and therefore this digital imaging can beadvantageously combined with image acquisition required for one or moreof the classification stages during this secondary classification step“E”.

For example, in one embodiment, after being removed from a developmentmedium in step “A”, and further being sorted and singulated in step “B”,during the embryo classification step “C”, the embryos may undergo twoclassification stages. First, a single-view (e.g., the top view)monochromatic image analysis is carried out to eliminate those embryosthat do not meet the basis size/shape criteria (block 46). Second, aclassification model is applied to the same single-view monochromaticimage to eliminate those embryos that are not likely to germinate (block48). In step “D”, those remaining embryos that have passed both of thetwo classification stages are placed in a tray and dried. Thereafter,during the secondary classification step “E”, the embryos forwarded fromstep “D” undergo a further series of classification stages that areperhaps more sophisticated and therefore time-consuming. For example,the embryos may be subjected to a multiple-view (e.g., the top view,side view, and end view) color image analysis to eliminate undesirableembryos according to a classification model (block 54), and further toan apical dome analysis (block 56) and/or a spectroscopic analysis(block 58) to still further eliminate undesirable embryos, againaccording to a suitable classification model.

FIG. 1B illustrates an alternative embodiment of a system for automatedharvesting and multi-stage screening of plant embryos. As with theembodiment of FIG. 1A, embryos are washed off a development surface andplaced onto the porous conveyor belt 24 and sieved, perhaps as assistedby additional washing with aqueous liquid (corresponding to FIG. 2,steps “A” and “B”). The embryos remaining on the conveyor belt 24 arethen imaged by a camera 38. The image of each embryo is transmitted tothe computer 40 to be analyzed and classified according to theirmorphological features (corresponding to FIG. 2, step “C”). For example,a suitable algorithm is used to identify those embryos that meet basicsize and shape criteria to be incorporated into manufactured seeds.

Then, a smart mini-robotic transfer system 60 under the control of thecomputer 40 is used to pick up and place each of those embryos meetingthe basic size and shape criteria onto a receiving tray 54 in an evenlyspaced array. Briefly, the transfer system 60 includes a housing 61laterally movable along a rail 62, and a robotic arm 63 extending fromthe housing 61 and including a vacuum tip end. The robotic arm 63 islongitudinally extendible and also axially rotatable. The details of oneexample of the mini-robotic transfer system 60 suitable for use in thepresent embodiment are disclosed in PCT Application Serial No.PCT/US00/40720 (WO 01/13702 A2) incorporated by reference above. In theillustrated embodiment of FIG. 1B, after an embryo is picked up from theconveyor belt 24 by the arm 63, the housing 61 is translated along therail 62 to a new position 61′, at which point the arm 63′ is extendeddownwardly to place the embryo on the tray 54. At this point, the arm61′ may be controllably rotated axially, based on the originalorientation of the embryo as imaged by the camera 38 and stored in thecomputer 40, so that the embryos as placed on the tray 54 are properlyoriented, for example, with their cotyledon ends all facing the samedirection. In one preferred embodiment, the embryos are precisely placedon the tray 54 in the same orientation and with their cotyledon endsprecisely aligned with each other. In the present description, a tray onwhich embryos are arranged in the same orientation and in a precisearray (e.g., with the positions of their cotyledon ends precisely known)is called an “index tray.”

Thereafter, the index trays 54 are forwarded to receive maturationtreatments, for example to the post development treatment room (dryingroom in the illustrated embodiment) 14 to dehydrate the embryos(corresponding to FIG. 2, step “D”). Next, the embryos may be rehydratedand undergo a secondary classification process (corresponding to FIG. 2,step “E”). Specifically, the index trays 54 each carrying a properlyoriented and evenly spaced array of embryos may be placed on a secondaryclassification conveyor belt 64, and the embryos may be subjected toadditional classification stages as they are transported on the conveyorbelt 64. For example, a suitable scanner 68, coupled to a computer 70,is used to further classify the embryos to identify those that arelikely to successfully germinate and grow into normal plants. During thesecondary classification, the use of the index tray 54 may beadvantageous because it permits localized analysis of each embryo on thetray. For example, certain imaging or spectroscopic analysis may becarried out with respect to a localized area of each embryo (e.g., itscotyledon end portion). Because the precise positions of the embryos(e.g., their cotyledon ends) on the index tray 54 are known, suchlocalized analysis is possible.

At the end of the secondary classification conveyor belt 64, anotherrobotic embryo placement system 71 is provided to pick up only thoseembryos that have been further selected as desirable, and to insert theminto manufactured seeds 76. In the illustrated embodiment, the embryoplacement system 71 includes a housing 72 translated along a rail 73 anda robotic arm 74 extending from the housing 72. After a desirable embryois picked up by the arm 74, the housing 72 is translated along the rail73 to a new position 72′, at which point the arm 74′ may be lowered toplace the embryo into a manufactured seed 76 (or a tubular restraint ofthe manufactured seed). The details of a suitable embryo placementsystem is disclosed in PCT Application Serial No. PCT/US00/40720 (WO01/13702 A2) discussed above. Various other alternative systems fortransferring and inserting the embryos into manufactured seeds 76 arepossible, as will be apparent to one skilled in the art. For example,the housing 72 and the arm 74 may be two- or three-dimensionallymovable. Also, a tray holding the plurality of manufactured seeds 76 maybe made one-, two-, or three-dimensionally movable so as to preciselyposition each of the seeds 76 relative to an embryo carried by theembryo placement system 71.

Notably, because the precise positions of the embryos on the index tray54 are known, the embryo placement system 71 needs not have thecapability to determine or correct the position and/or orientation ofeach embryo as it is picked up from the tray 54. For example, based onthe known position and orientation of each embryo, it is possible forthe embryo placement system 71 to precisely position the cotyledon endof each embryo within the manufactured seed 76.

According to the invention, a complete method and system forautomatically harvesting somatic embryos are provided, which couldreplace the current manual operation including the steps of sorting andsingulating and further classifying mass-produced embryos according totheir putative germination vigor. Classification of the embryos iscarried out in multiple stages to efficiently identify those embryosthat are suited for incorporation into manufactured seeds. By carefullyselecting suitable classification methods to be combined together, onecan achieve progressively higher selection accuracy that would match orexceed the level of selectivity currently achievable only by a highlyskilled technician. Further, the throughput of the present automatedmethod of multi-stage screening (classification) is calculated to beapproximately 5 million embryos per year, which is sufficient to meetthe 1.5-2 seconds/embryo rate required for the classification of sortedand singulated embryos for the purpose of mass production ofmanufactured seeds.

While the preferred embodiments of the invention have been illustratedand described, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of automatically harvesting and screening plant embryos inmultiple stages, comprising: automatically sorting and singulating plantembryos; automatically classifying the sorted and singulated plantembryos using a first classification method; and automaticallyclassifying the plant embryos that have passed the first classificationmethod using a second classification method.
 2. The method of claim 1,wherein the step of automatic sorting and singulating comprisesautomatically removing plant embryos from a development medium.
 3. Themethod of claim 2, wherein the automatic removal of plant embryoscomprises washing off the plant embryos from the development mediumusing aqueous liquid.
 4. The method of claim 1, wherein the step ofsorting and singulating embryos comprises using a flow cytometer.
 5. Themethod of claim 1, wherein the step of sorting and singulating embryoscomprises placing the embryos onto a porous material for sieving.
 6. Themethod of claim 5, wherein the embryos placed on the porous material aresprayed with aqueous liquid to remove any suspensor tissues or residualembryonal suspensor masses (ESM) from the embryos.
 7. The method ofclaim 5, wherein the porous material comprises a porous conveyor belt.8. The method of claim 7, wherein the embryos are placed in a reservoirwhich dispense the embryos onto the porous conveyor belt, the reservoirbeing configured to dispense the embryos onto the belt in a regulatedmanner.
 9. The method of claim 8, wherein the reservoir comprises apipe.
 10. The method of claim 7, wherein the plant embryos sorted andsingulated after sieving are placed on a classification conveyor belt,along which the first and second automatic classification methods arecarried out.
 11. The method of claim 10, wherein the sorted andsingulated plant embryos are transferred from the porous conveyor beltto the classification conveyor belt by gravity.
 12. The method of claim10, wherein the classification conveyor belt extends generallyperpendicularly to the porous conveyor belt.
 13. The method of claim 10,wherein the sorted and singulated plant embryos are placed on theclassification conveyor belt in a single file along the length of theclassification conveyor belt.
 14. The method of claim 13, wherein theplant embryos are placed in a single file spaced apart from each other.15. The method of claim 10, further comprising the step of automaticallyremoving those embryos that have been classified as undesirable by thefirst or second automatic classification method from the classificationconveyor belt.
 16. The method of claim 15, wherein the automatic removalof undesirable embryos is carried out by a jet.
 17. The method of claim10, further comprising the step of automatically removing those embryosthat have been classified as desirable by the first and second automaticclassification methods from the classification conveyor belt.
 18. Themethod of claim 17, wherein the step of automatically removing desirableembryos comprises transferring those desirable embryos onto a receivingsurface in a predefined array.
 19. The method of claim 18, wherein thereceiving surface comprises a tray mounted on a motorized platformconfigured to adjust the position of the tray relative to theclassification conveyor belt so as to receive the embryos dropping bygravity from the classification conveyor belt into a predefined array.20. The method of claim 19, wherein the dropping of the embryos bygravity from the classification conveyor belt is assisted by a jet. 21.The method of claim 7, wherein the sorted and singulated plant embryosare classified according to the first classification method while on theporous conveyor belt, and the plant embryos that have passed the firstclassification method are transferred from the porous conveyor belt ontoan index tray in a predefined array.
 22. The method of claim 21, whereinthe second classification method is carried out on the plant embryosthat are placed on the index tray.
 23. The method of claim 1, whereinthe first classification step classifies the embryos based on theirshape and size and the second classification step classifies the embryosbased on their putative germination vigor according to a predefinedclassification model.
 24. The method of claim 1, wherein the secondclassification method is more selective and time-consuming than thefirst classification method.
 25. A system for automatically harvestingand screening plant embryos in multiple stages, comprising: means forautomatically sorting and singulating plant embryos; means forautomatically classifying the sorted and singulated plant embryos usinga first classification method; and means for automatically classifyingthe plant embryos that have passed the first classification method usinga second classification method.